The differentiation of specific cell types in mammals is mediated by differential gene expression. To understand the regulation of the development and differentiation of a particular cell type, it is important to understand what differential gene expression occurs in that cell type and how this gene expression is regulated. For the last 12 years we have been studying one gene (phosphoglycerate kinase-2 or Pgk-2) that is specifically expressed in spermatogenic cells in mammals. In that time, we have learned a great deal about the expression, regulation, structure, function and evolution of this gene. In this application, we propose additional experiments to help us further understand the how the spermatogenesis-specific expression of the Pgk-2 is regulated at the level of transcription. We believe that by learning more about how the expression of this single gene is regulated in these cells, we will gain insight into how many of the genes that are differentially expressed in spermatogenic cells might be regulated. In this way, we hope to contribute to a better understanding of how the process of spermatogenesis is, itself, regulated. Our emphasis in this application is to study the developmental order of molecular events that predispose the initiation of transcription of the Pgk-2 gene in spermatogenic cells, and also to follow the "reprogramming" of these events following cessation of Pgk-2 transcription. The specific aims of this application include an investigation of the occurrence of protein-DNA interactions in the promoter region of the Pgk-2 gene in spermatogenic cells in vivo, a molecular characterization of testis-specific nuclear transcription factors that are critically involved in the tissue-specific regulation of transcription of the Pgk-2 gene, and a developmental study of changes in chromatin structure, association of the Pgk-2 gene with the nuclear matrix, and timing of its replication that predispose and follow active transcription of this gene. We believe that by learning more about the order of the various molecular events that regulate Pgk-2 transcription, we will gain a much better understanding of die overall mechanism of gene regulation in spermatogenic cells. This could provide valuable insight into avenues of treatment for defective gene expression during spermatogenesis, and/or other genetic manipulations involving these cells.